Effect of spermidine on ameliorating spermatogenic disorders in diabetic mice via regulating glycolysis pathway

Abstract

Diabetes mellitus (DM), a high incidence metabolic disease, is related to the impairment of male spermatogenic function. Spermidine (SPM), one of the biogenic amines, was identified from human seminal plasma and believed to have multiple pharmacological functions. However, there exists little evidence that reported SPM's effects on moderating diabetic male spermatogenic function. Thus, the objective of this study was to investigate the SPM's protective effects on testicular spermatogenic function in streptozotocin (STZ)-induced type 1 diabetic mice. Therefore, 40 mature male C57BL/6 J mice were divided into four main groups: the control group (n = 10), the diabetic group (n = 10), the 2.5 mg/kg SPM-treated diabetic group (n = 10) and the 5 mg/kg SPM-treated diabetic group (n = 10), which was given intraperitoneally for 8 weeks. The type 1 diabetic mice model was established by a single intraperitoneal injection of STZ 120 mg/kg. The results showed that, compare to the control group, the body and testis weight, as well the number of sperm were decreased, while the rate of sperm malformation was significantly increased in STZ-induced diabetic mice. Then the testicular morphology was observed, which showed that seminiferous tubule of testis were arranged in mess, the area and diameter of which was decreased, along with downregulated anti-apoptotic factor (Bcl-2) expression, and upregulated pro-apoptotic factor (Bax) expression in the testes. Furthermore, testicular genetic expression levels of Sertoli cells (SCs) markers (WT1, GATA4 and Vimentin) detected that the pathological changes aggravated observably, such as the severity of tubule degeneration increased. Compared to the saline-treated DM mice, SPM treatment markedly improved testicular function, with an increment in the body and testis weight as well as sperm count. Pro-apoptotic factor (Bax) was down-regulated expression with the up-regulated expression of Bcl-2 and suppression of apoptosis in the testes. What's more, expression of WT1, GATA4, Vimentin and the expressions of glycolytic rate-limiting enzyme genes (HK2, PKM2, LDHA) in diabetic testes were also upregulated by SPM supplement. The evidence derived from this study indicated that the SMP's positive effect on moderating spermatogenic disorder in T1DM mice's testis. This positive effect is delivered via promoting spermatogenic cell proliferation and participating in the glycolytic pathway's activation.

Keywords: Diabetes; Glycolytic pathway; Sertoli cells; Spermatogenic dysfunction; Spermidine.

Fig. 1

Effects of spermidine on the morphological structure of epididymis and testis in diabetic mice. A HE staining of epididymis and testes. B The area of seminiferous tube. C The diameter of seminiferous tube. D The eosin-Y staining of sperms. (1) and (2) are the Ctrl group sperms, (3) (4) and (5) are the DM group sperms, (6) and (7) are the SPM 2.5 mg/kg group sperms, (8) and (9) are the SPM 5 mg/kg group sperms

Fig. 2

Effects of spermidine on apoptosis of spermatogenic cells in diabetic mics. The red arrows indicate the cells with positive signal. A (1) The expression of Bcl-2 in testis detected by qRT-PCR. (2) The expression of Bax in testis detected by qRT-PCR. (3) The expression of Bcl-2/Bax in testis detected by qRT-PCR. B The expression of Bcl-2 in spermatogenic tubules was detected by immunohistochemistry. C The expression of Bax in spermatogenic tubules was detected by immunohistochemistry. D:(1) Bcl-2 positive cell count in the testis. (2) Bax positive cell count in the testis

Fig. 3

Effects of spermidine on the number and function of testicular Sertoli cells in diabetic mice. The red arrows indicate the cells with positive signal. A (1) The expression of GATA4 in testis detected by qRT-PCR. (2) The expression of WT1 in testis detected by qRT-PCR. B The expression of Vimentin in spermatogenic tubules was detected by immunohistochemistry. C The expression of Vimentin in spermatogenic tubules was detected by immunohistochemistry

Fig. 4

Effects of spermidine glycolytic rate-limiting enzyme gene in diabetic mics testis. A (1) The expression of HK2 in testis detected by qRT-PCR. (2) The expression of PKM2 in testis detected by qRT-PCR. (3) The expression of LDHA in testis detected by qRT-PCR. B HK2, PKM2, LDHA protein levels in spermatogenic tubules was detected by Western Blot. NS: normal saline

Fig. 5

Effects of spermidine glycolytic rate-limiting enzyme gene in diabetic mics testis. The red arrows indicate the cells with positive signal. A The expression of HK2 in spermatogenic tubules was detected by immunohistochemistry. B The expression of PKM2 in spermatogenic tubules was detected by immunohistochemistry. C The expression of LDHA in spermatogenic tubules was detected by immunohistochemistry. D (1) HK2 positive cell count in the testis. (2) PKM2 positive cell count in the testis. (3) LDHA positive cell count in the testis

Fig. 6

In vivo supplementation of the spermidine (SPM) effectively protective DM-induced male reproductive damage by improving the pathological structure of testis, inhibiting apoptosis of spermatogenic cells and promoting proliferation. Eventually, SPM could ameliorate the structure and function of SCs in DM mice, increase the expression of glycolytic rate-limiting enzyme